Secreted soluble alpha2delta-2, alpha2delta-3, or alpha2delta-4 calcium channel subunit polypeptides and screening assays using same

ABSTRACT

Soluble α 2 δ sub-type polypeptides.  
     Methods for cloning, expression and purification of freely soluble α 2 δsubtype polypeptides.  
     A method for the screening of ligands which bind to soluble α 2 δ subtype polypeptides.

BACKGROUND OF THE INVENTION

Voltage-dependent Ca²⁺ channels (VDCCs) are heteromultimeric complexespresent in both neuronal and non-neuronal tissues, including heart andskeletal muscle. VDCCs are minimally composed of three subunits: apore-forming transmembrane α₁ subunit, a hydrophilic intracellular βsubunit, and a membrane-associated α₂δ subunit; a transmembrane γsubunit is also found in skeletal muscle tissue. Multiple subtypesand/or splice variants of the α₁, β, and α₂δ subunits have been found.

Gabapentin ((1-aminomethyl)cyclohexane acetic acid or Neurontin) is astructural analogue of GABA, which is mainly used as an adjunctivetherapy for epilepsy. Recent research suggests that gabapentin may alsohave clinical utility for various indications including anxiety andpain. Although designed as a lipophilic GABA-mimetic, gabapentin doesnot have a high affinity for either GABA_(A) or GABA_(B) receptors, GABAuptake sites, or the GABA-degrading enzyme GABA-transaminase (EC2.6.1.19).

A novel high affinity binding site for [³H]gabapentin in rat, mouse, andporcin brains has been characterized. Recently, the[³H]gabapentin-binding protein was isolated from pig brain andidentified as the α₂δ-1 subunit of VDCCs. None of the prototypicanticonvulsant drugs displace [³]gabapentin binding from the α₂δ-1subunit. [³H]Gabapentin-binding is stereospecifically inhibited by twoenantiomers of 3-isobutyl GABA. The rank order of potency of gabapentin,and S- and R-isobutyl GABA, at the [³H]gabapentin binding site mirrorstheir anticonvulsant activity in mice. However, electrophysiologicalstudies have yielded conflicting data on the action of gabapentin atVDCCs.

The α₂δ subunit is derived from a single gene, the product of which isextensively post-translationally modified particularly through thecleavage of the signal sequence. The polypeptide is cleaved to formdisulfide-bridged α₂ and δ peptides, both of which are heavilyglycosylated. Although it seems clear today that the α₂ and δ peptidesare membrane-associated peptides, it is unclear whether these peptidescomprise one or several transmembrane domains. Furthermore, thelocation, size and structural configuration of these eventualtransmembrane domains remains to be determined.

But in any event, the fact that α₂δ is a membrane-associated protein,regardless of its precise structural configuration, renders its largescale expression in recombinant systems difficult. Indeed, as the α₂δprotein is targeted to the membrane, it requires detergentsolubilisation to release it for purification. This important drawbackimposes considerable restrictions for any potential applicationsrequiring large amounts of recombinant protein. Furthermore, the varioussubtypes of α₂δ subunits are different proteins with very lowhomologies. It is therefore extremely difficult to predict theirrespective behaviors, for example in gene truncation experiments.

The only assay currently available for the screening of ligands thatbind the α₂δ subunit involves the use of pig membrane extracts as asource of the α₂δ subunit. Such an assay presents major inconvenients.Firstly, bcause the assay material is a membrane extract, it is verydifficult to accurately determine the protein composition from one assaypreparation to another particularly with regard to the subtype. Also,the presence of various impurities in the assay preparation is a problemin small plate assays. Furthermore, as the protein preparation lackshomogeneity, the interaction between the targeted protein and the assayplate is often quite uneven. This rendres the streamlining of the assayin a high throughput format almost impossible to achieve.

SUMMARY OF THE INVENTION

In the context of the present invention, the inventors have found thatit was possible to delete a portion of the nucleotide sequence encodinga eukaryotic, preferably a mammal cerebral cortical voltage-dependentcalcium channel α₂ subunit to yield a soluble secreted protein whichretains its affinity for [³H]gabapentin.

The invention concerns:

1) A purified or isolated nucleic acid encoding a mammalian secretedsoluble cerebral cortical voltage-dependent calcium channel α₂δ-2, α₂δ-3or α₂δ-4 subunit polypeptide.

2) A purified or isolated nucleic acid according to 1), comprising apolynucleotide having at least 90% identity with the sequence encoding:

-   -   from amino-acid 1 to between amino-acids 1027 and 1062 of SEQ ID        No 20 for α₂δ-2,    -   from amino-acid 1 to between amino-acids 984 and 1019 of SEQ ID        No 22 for α₂δ-3.

3) A purified or isolated nucleic acid according to 1), having at least90% identity with the sequence encoding:

-   -   from amino-acid 1 to between amino-acids 1047 and 1062 of SEQ ID        No 20 for α₂δ-2,    -   from amino-acid 1 to between amino-acids 1004 and 1019 of SEQ ID        No 22 for α₂δ-3.

4) A purified or isolated nucleotide sequence according to 1) whereinsaid sequence is the sequence of SEQ ID No1, SEQ ID No2, SEQ ID No3, SEQID No7, SEQ ID No8, SEQ ID No9, SEQ ID No13, SEQ ID No14, SEQ ID No15,SEQ ID No19 or SEQ ID No21.

5) A purified or isolated nucleic acid, having at least 90% identitywith the nucleotide sequence of SEQ ID No19 or SEQ ID No21.

6) A purified or isolated polynucleotide comprising at least 10consecutive nucleotides of the nucleotide sequence of SEQ ID No19 or SEQID No21.

7) A polynucleotide probe or primer hybridizing, under stringentconditions, with the with the nucleotide sequence of SEQ ID No19 or SEQID No21.

8) A method for the amplification of a nucleic acid encoding a mammaliansecreted soluble cerebral cortical voltage-dependent calcium channelα₂δ-n subunit polypeptide wherein n is 2, 3 or 4, said method comprisingthe steps of:

-   -   (a) contacting a test sample suspected of containing the target        secreted soluble α₂δ-n subunit nucleic acid, or a sequence        complementary thereto, with an amplification reaction reagent        comprising a pair of amplification primers located on either        side of the α₂δ-n subunit nucleic acid region to be amplified,        and    -   (b) optionally, detecting the amplification products.

9) A kit for the amplification of a nucleic acid encoding a secretedsoluble α₂δ-n subunit polypeptide wherein n is 2, 3 or 4, or acomplementary sequence thereto in a test sample, wherein said kitcomprises:

-   -   (a) a pair of oligonucleotide primers which can hybridize, under        stringent conditions, to the secreted soluble α₂δ-n subunit        nucleic acid region to be amplified;    -   (b) optionally, the reagents necessary for performing the        amplification reaction.

10) A recombinant vector comprising a nucleic acid according to 1).

11) A recombinant host cell comprising a nucleic acid according to 1).

12) A method for producing a secreted soluble α₂δ-n subunit wherein n is2, 3 or 4, and said method comprises the steps of:

-   -   (a) inserting the nucleic acid encoding the desired α₂δ-n        subunit polypeptide in an appropriate vector;    -   (b) culturing, in an appropriate culture medium, a host cell        previously transformed or transfected with the recombinant        vector of step (a);    -   (c) harvesting the culture medium thus obtained or lyse the host        cell, for example by sonication or osmotic shock;    -   (d) separating or purifying, from said culture medium, or from        the pellet of the resultant host cell lysate, the thus produced        α₂δ-n subunit polypeptide of interest.

13) A purified or isolated recombinant polypeptide comprising the aminoacid sequence of a secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide.

14) A recombinant polypeptide according to 14), having at least 80%amino-acid identity with a polypeptide comprising:

-   -   from amino acid 1 to between amino acids 1027 and 1062 of the        amino acid sequence of SEQ ID No20, or    -   from amino acid 1 to between amino acids 1019 and 1079 of the        amino acid sequence of SEQ ID No22.

15) A recombinant polypeptide according to 14), wherein said recombinantpolypeptide is selected from the group consisting of the amino acidsequences of SEQ ID no4, SEQ ID no5, SEQ ID no6, SEQ ID no10, SEQ IDno11, SEQ ID no12, SEQ ID no16, SEQ ID no17 and SEQ ID no18.

16) A method for the screening of ligands which bind a cerebral corticalvoltage-dependent calcium channel α₂δ-n subunit wherein n is 2, 3 or 4,said method comprising the steps of:

-   -   contacting a secreted soluble recombinant calcium channel α₂δ-n        subunit polypeptide with:        -   a ligand of interest; and        -   a labelled compound which binds the α₂δ-n subunit; and    -   measuring the level of binding of the labelled compound to the        α₂δ-n subunit.

17) A method according to 16), wherein said method is a scintillationproximity assay.

18) A method according to 16), wherein said method is a flashplateassay.

19) A method according to 16), wherein said method is a filter bindingassay.

20) A method according to 16), wherein said secreted soluble recombinantcalcium channel α₂δ-n subunit polypeptide is selected from polypeptideshaving at least 80%, preferably 90%, more preferably 95%, and mostpreferably 98 or 99% amino-acid identity with the polypeptide comprisingfrom amino acid 1 to between amino-acids 984 and 1063, preferablybetween amino-acids 994 and 1054, and most preferably betweenamino-acids 1019 and 1054 of SEQ ID No5 or SEQ ID No16.

21) A method according to 16), wherein said secreted soluble recombinantcalcium channel α₂δ-n subunit polypeptide is selected from the groupconsisting of SEQ ID No6, 7, 8, 9, 13, 14 and 15, with the polypeptidesof SEQ ID No9 and SEQ ID No15 being most preferred.

22) A kit for the screening of ligands which bind bind a cerebralcortical voltage-dependent calcium channel α₂δ-n subunit wherein n is 2,3 or 4, said kit comprising:

-   -   a secreted soluble recombinant calcium channel α₂δ-n subunit;        and    -   a labelled compound which binds to the α₂δ-n subunit.

Hence, the invention concerns nucleotide sequence fragments of acerebral cortical voltage dependent calcium channel α₂δ-2, α₂δ-3 orα₂δ-4 subunit cDNA encoding a soluble secreted α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide(hereinafter a α₂δ-2, α₂δ-3 or α₂δ-4 subunit).Preferably, these nucleotide sequences encode a soluble secreted α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide bearing a gabapentin or a[³H]gabapentin binding site. More preferably, the soluble secretedα₂δ-2, α₂δ-3 or α₂δ-4 subunit nucleic acid is derived from a eukaryotic,preferably a mammal, more preferably a human α₂δ-2, α₂δ-3 or α₂δ-4subunit.

A further object of the present invention concerns recombinant vectorscomprising a nucleic acid sequence encoding a soluble secreted α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide.

The invention also encompasses host cells and transgenic non-humanmammals comprising said nucleic acid sequences or recombinant vectors.

The invention also concerns a soluble secreted α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide which is characterized in that it is a solublesecreted polypeptide having affinity for [³H]gabapentin. Preferably, thesoluble secreted polypeptide is derived from a mammal, more preferably ahuman α₂δ-2, α₂δ-3 or α₂δ-4 subunit.

The inventors have also found that it was possible to use a solublesecreted form of a voltage-dependant calcium channel α₂δ-2, α₂δ-3 orα₂δ-4 subunit polypeptide in an assay for the screening of ligands whichbind the α₂δ-2, α₂δ-3 or α₂δ-4 subunit.

The invention therefore also concerns a method for the screening ofligands which bind a calcium channel α₂δ-2, α₂δ-3 or α₂δ-4 subunit.

The method comprises the steps of:

-   -   contacting a secreted soluble recombinant calcium channel α₂δ-2,        α₂δ-3 or α₂δ-4 subunit polypeptide with:        -   a ligand of interest; and        -   a labelled compound which binds a α₂δ-2, α₂δ-3 or α₂δ-4            subunit; and    -   measuring the level of binding of the labelled compound to the        secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit.

The invention also concerns a kit for the screening of ligands whichbind a calcium channel α₂δ-2, α₂δ-3 or α₂δ-4 subunit.

The kit comprises:

-   -   a secreted soluble recombinant calcium channel α₂δ-2, α₂δ-3 or        α₂δ-4 subunit polypeptide; and    -   a labelled compound which binds a calcium channel α₂δ-2, α₂δ-3        or α₂δ-4 subunit.

DETAILED DESCRIPTION OF TIRE INVENTION

The invention concerns truncated α₂δ-2, α₂δ-3 or α₂δ-4 subunit cDNAsequences. These truncated sequences encode soluble secretedpolypeptides which retain their affinity for [³M]gabapentin.

Throughout the present specification, the expression “nucleotidesequence” is used to designate indifferently a polynucleotide or anucleic acid. More precisely, the expression “nucleotide sequence”encompasses the nucleic material and the sequence information and is notrestricted to the sequence information (i.e. the succession of letterschosen among the four base letters) that biochemically characterizes aspecific DNA or RNA molecule.

As used interchangeably herein, the terms “oligonucleotides”, “nucleicacids” and “polynucleotides” include RNA, DNA, or RNA/DNA hybridsequences of more than one nucleotide in either single chain or duplexform.

Further to its general meaning understood by the one skilled in the art,the term “nucleotide” is also used herein to encompass modifiednucleotides which comprise at least one of the following modifications(a) an alternative linking group,.(b) an analogous form of purine, (c)an analogous form of pyrimidine, or (d) an analogous sugar. For examplesof analogous linking groups, purines, pyrimidines, and sugars, see forexample PCT publication NoWO 95/04064.

The polynucleotide sequences of the invention may be prepared by anyknow method, including synthetic, recombinant, or a combination thereofas well as through any purification methods known in the art.

A) Secreted α₂δ-2, α₂δ-3 or α₂δ-4 Subunit Polypeptides

The invention comprises polynucleotide sequences encoding a solublesecreted eukaryotic, preferably a soluble secreted mammal α₂δ-2, α₂δ-3or α₂δ-4 subunit polypeptide. These sequences particularly include butare not restricted to 1) those sequences encoding a soluble secretedpolypeptide of this α₂δ-2, α₂δ-3 or α₂δ-4 subunit which preferablyretains its binding affinity for [³H]gabapentin and 2) nucleotidefragments useful as nucleic acid primers or probes for amplification ordetection purposes.

The expression “soluble secreted α₂δ-2, α₂δ-3 or α₂δ-4 subunit” isintended to designate polypeptide sequences which, when produced by arecombinant host cell, are secreted at least partially into the culturemedium rather than remaining associated with the host cell membrane.

1) cDNA Fragments Encoding Soluble Secreted α₂δ-2, α₂δ-3 or α₂δ-4Subunit Polypeptides

One of the important embodiments of the present invention concernstruncated nucleotide sequences of α₂δ-2, α₂δ-3 or α₂δ-4 subunit cDNAswhich encode soluble secreted α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptides. The inventors have found that it was possible to generatedeletion mutants of α₂δ-2, α₂δ-3 or α₂δ-4 subunit cDNAs which, whenexpressed, produce a significant amount of soluble secreted proteins,preferably soluble secreted proteins, which retain their [³H]gabapentinbinding affinity. These truncated nucleotide sequences of the inventionare of significant value to the skilled person as they now allow fastand reliable access to significant concentrations of selected solublesecreted α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptides. To that end, theinventors have determined the minimal and optimal fragment lengthsrequired to express a polypeptide which: 1) binds [³H]gabapentin withsufficient affinity and; 2) is obtained in a soluble secreted form.

The discussion provided below provides comments on possible truncations,giving as an example the human α₂δ-2, α₂δ-3 or α₂δ-4 subunit. However,given the very substantial cross-species homology for α₂δ-2, α₂δ-3 orα₂δ-4 subunit sequences, the comments below can also be applied to othereukaryotic species, and more particularly other mammalian species suchas rat, mouse, rabbit or pig. Their α₂δ-2, α₂δ-3 or α₂δ-4 subunitsequences, which for most are available in public databases, share avery substantial homology with the human α₂δ-2, α₂δ-3 or α₂δ-4 subunitsequences.

The inventors believe that the soluble secreted α₂δ-2 subunitpolypeptides which are as close as possible to the native sequence andwhich are therefore more likely to retain their native folding and hencetheir [³1H]gabapentin binding properties are those corresponding to thenative protein in which amino-acid stretch _(—)1027_ to _the C-terminalend_ of the amino-acid sequence of SEQ ID No20 has been deleted. Theskilled scientist can quite easily determine within this amino-acidstretch the optimal α₂δ-2 subunit polypeptides.

The inventors also believe that the soluble secreted α₂δ-3subunit-polypeptides which are as close as possible to the nativesequence and which are therefore more likely to retain their nativefolding and hence thir [³H]gabapentin binding properties are thosecorresponding to the native protein in which amino-acid stretch _(—)984_to _C-terminal end_ of the amino-acid sequence of SEQ ID No22 has beendeleted. The skilled scientist can quite easily determine within thisamino-acid stretch the optimal α₂δ-3 subunit polypeptides.

The invention therefore particularly concerns a nucleotide sequenceencoding a polypeptide having at least 80% identity with the polypeptidecomprising from amino-acid 1 to between amino-acids _(—)1027_ and_(—)1145_, preferably to between amino-acids _(—)1062_ and _(—)1145 ofSEQ ID No20.

Preferred nucleotide sequences include those of SEQ ID No1, SEQ ID No 2and SEQ ID No3.

The invention also concerns a nucleotide sequence encoding a polypeptidehaving at least 80% identity with the polypeptide comprising fromamino-acid 1 to between amino-acids _(—)984_ and 1085_, preferably tobetween amino-acids 1019_ and _(—)1085 of SEQ ID No22.

Preferred nucleotide sequences include those of SEQ ID No7, SEQ ID No 8and SEQ ID No9.

B) Amplification of the Soluble Secreted α₂δ-2, α₂δ-3 or α₂δ-4 SubunitNucleotide Sequences

Another object of the invention consists of a method for theamplification of a nucleic acid encoding a soluble secreted α₂δ-2, α₂δ-3or α₂δ-4 subunit polypeptide, preferably a polypeptide bearing a[³H]gabapentin binding site, said method comprising the steps of:

-   -   (a) contacting a test sample suspected of containing the target        α₂δ-2, α₂δ-3 or α₂δ-4 subunit nucleic acid, a fragment or a        variant thereof, or a sequence complementary thereto, with an        amplification reaction reagent comprising a pair of        amplification primers which can hybridize under stringent        conditions, the α₂δ-2, α₂δ-3 or α₂δ-4 subunit nucleic acid        region to be amplified, and    -   (b) optionally, detecting the amplification products.

The expression [³H]gabapentin binding site, when used herein is intentedto designate a site which can bind either [³H]gabapentin or otherligands such as (S+)-3-isobutyl gaba or (R−)-3-isobutyl gaba.

In a first preferred embodiment of the above method, the nucleic acidencodes a secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide ofSEQ ID No4, SEQ ID No5, SEQ ID No6, SEQ ID No10, SEQ ID No11, SEQ IDNo12, SEQ ID No16, SEQ ID No17 and SEQ ID no18.

In a second preferred embodiment of the above amplification method, theamplification product is detected by hybridization with a labelled probehaving a sequence which is complementary to the amplified region.

C) Recombinant Vectors and Hosts Cells for the Expression of a SecretedSoluble α₂δ-2, α₂δ-3 or α₂-4 Subunit Polypeptide

1) Recombinant Vectors

The present invention also encompasses a family of recombinant vectorscomprising any one of the nucleic acids described herein. Firstly, theinvention deals with a recombinant vector comprising a nucleic acidselected from the group consisting of:

-   -   (a) a purified or isolated nucleic acid encoding a a secreted        soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit having at least 80% amino        acid identity with the polypeptide of SEQ ID No20 or 22, or a        sequence complementary thereto;    -   (b) a purified or isolated nucleic acid having at least 90%        nucleotide identity with a polynucleotide selected from the        group consisting of the nucleotide sequences of SEQ ID No1, SEQ        ID No2, SEQ ID No3, SEQ ID No 7, SEQ ID No8, SEQ ID No9, SEQ ID        No 13, SEQ ID No14, SEQ ID No15 or a sequence complementary        thereto;    -   (c) a purified or isolated polynucleotide comprising at least 10        consecutive nucleotides of a nucleic acid described in (a)        or (b) or a sequence complementary thereto.

In a first preferred embodiment a recombinant vector of the invention isused to amplify the inserted polynucleotide of the invention in asuitable host cell, this polynucleotide being amplified every time therecombinant vector replicates.

Recombinant expression vectors comprising a nucleic acid encodingsecreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptides that aredescribed in the present specification are also part of the invention.These include, but are not restricted to, nucleic acids encoding fromamino-acid 1 to between amino-acids 1027 and 1145, preferably betweenamino-acids 1062 and 1145 of SEQ ID No20, as well as nucleic acidsencoding from amino-acid 1 to between amino-acids 984 and 1085,preferably between amino-acids 1019 and 1085, of SEQ ID No22.

Another preferred embodiment of the recombinant vectors according to theinvention consist of expression vectors comprising a nucleic acidencoding α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptides of the invention,and more preferably a nucleic acid encoding a polypeptide selected fromthe group consisting of the amino acid sequences of SEQ ID No4, SEQ IDNo5, SEQ ID No6, SEQ ID No10, SEQ ID No11, SEQ ID No12, SEQ ID No16, SEQID No17 and SEQ ID no18.

Within certain embodiments, expression vectors can be employed toexpress the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptideswhich can then be purified and for example, be used as a immunogen inorder to raise specific antibodies directed against said secretedsoluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptides.

Preferred eukaryotic vectors of the invention are listed hereafter asillustrative but not limitative examples: pcDNA3, pFLAG, pCMV-Script,pIND, pMC1NEO, pHIL, pGAPZA, pMT/V5-His-TOPO, pMT/V5-His,pAc5.1/V5-HisA, pDS47/V5-His, pcDNA4, pcDNA6, pEF1, pEF4, pEF6, pUB6,pZeoSV2, pRc/CMv2, pcDM8, pCR3.1, pDisplay, pSecTag2, pVP22, pEMZ,pCMV/Zeo, pSinRep5, pCEP, pREP, pHook-1

Preferred bacteriophage recombinant vectors of the invention are P1bacteriophage vectors such as described by Sternberg N. L. (1992;1994).

A suitable vector for the expression of a soluble secreted α₂δ-2, α₂δ-3or α₂δ-4 subunit polypeptide is a baculovirus vector that can bepropagated in insect cells and in insect cell-lines. Specific suitablehost vectors includes, but are not restricted to :pFastBac-1,pIZ/V5-His, pBacMan-1, pBlueBac4.5, pBlueBacHis2, pMelBacA, pVL1392,pVL1393

The recombinant expression vectors from the invention may also bederived from an adenovirus such as those described by Feldman and Steig.(1996) or Ohno et al. (1994). Another preferred recombinant adenovirusaccording to this specific embodiment of the present invention is thehuman adenovirus type two or five (Ad 2 or Ad 5) or an adenovirus ofanimal origin (French Patent Application no FR 93 05 954).

a) Regulatory Expression Sequences

Expression requires that appropriate signals are provided in thevectors, said signals including various regulatory elements such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. The regulatorysequences of the expression vectors of the invention are operably linkedto the nucleic acid a soluble secreted α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or an enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence.

More precisely, two DNA molecules (such as a polynucleotide containing apromoter region and a polynucleotide encoding a desired polypeptide orpolynucleotide) are said to be “operably linked” if the nature of thelinkage between the two polynucleotides does not: (1) result in theintroduction of a frame-shift mutation or (2) interfere with the abilityof the polynucleotide containing the promoter to direct thetranscription of the coding polynucleotide.

Generally, recombinant expression vectors include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in an appropriate frame with thetranslation, initiation and termination sequences, and preferably aleader sequence capable of directing sequences of the translated proteininto the periplasmic space or the extra-cellular medium. In a specificembodiment wherein the vector is adapted for transfecting and expressingdesired sequences in eukaryotic host cells, preferred vectors comprisean origin of replication from the desired host, a suitable promoter andan enhancer, and also any necessary ribosome binding sites,polyadenylation site, transcriptional termination sequences, andoptionally 5′-flanking non-transcribed sequences.

DNA sequences derived from the SV 40 viral genome, for example SV 40origin early promoter, enhancer, and polyadenylation sites may be usedto provide the required non-transcribed genetic elements.

b) Promoter Sequences

Suitable promoter regions used in the expression vectors according tothe invention are chosen taking into account the host cell in which theheterologous nucleic acids have to be expressed.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression, or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed.

Additionally, the promoter is generally heterologous with respect to therecombinant vector sequences within which the construct promoter/codingsequence has been inserted. Preferred eukaryotic promoters are the CMV,polyhidran or OPIE2.

c) Recombinant Host Cells

Host cells that have been transformed or transfected with one of thenucleic acids described herein, or with one of the recombinant vector,particularly recombinant expression vector, described herein are alsopart of the present invention.

Are included host cells that are transformed (prokaryotic cells) or aretransfected (eukaryotic cells) with a recombinant vector such as one ofthose described above.

Preferred host cells used as recipients for the expression vectors ofthe invention are the following:

-   -   (a) prokaryotic host cells: Escherichia coli, strains. (i.e.        DH10 Bac strain) Bacillus subtilis, Salmonella typhimurium and        strains from species such as Pseudomonas, Streptomyces and        Staphylococcus;    -   (b) eukaryotic host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1;        NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL        1650; NoCRL 1651), Sf-9 cells (ATCC NoCRL 1711), C127 cells        (ATCC NoCRL-1804), 3T3 cells (ATCC NoCRL-6361), CHO cells (ATCC        NoCCL-61), human kidney 293 cells (ATCC No 45504; NoCRL-1573),        BHK (ECACC No84100 501; No84111301), sf 9, sf 21 and hi-5 cells.        D) Production of Recombinant Secreted Soluble α₂δ-2, α₂δ-3 or        α₂δ-4 Subunit Polypeptides

The present invention also concerns a method for producing one of theamino acid sequences described herein and especially a polypeptideselected from the group consisting of the aminoacid sequences of SEQ IDNo4, SEQ ID No5, SEQ ID No6, SEQ ID No10, SEQ ID no11, SEQ ID no12, SEQID no16, SEQ ID no17 or SEQ ID no18 wherein said method comprises thesteps of:

-   -   (a) inserting the nucleic acid encoding the desired amino acid        sequence in an appropriate vector;    -   (b) culturing, in an appropriate culture medium, a host cell        previously transformed or transfected with the recombinant        vector of step (a);    -   (c) harvesting the culture medium thus obtained or lyse the host        cell, for example by sonication or osmotic shock;    -   (d) separating or purifying, from said culture medium, or from        the pellet of the resultant host cell lysate, the thus produced        recombinant polypeptide of interest.

In some instances, it is required to tag the secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide prior to purification. The tag isthen in most instances encoded into the nucleotide sequence that is needto express the polypeptide. Examples of such tags include, but are notlimited to sequences encoding C-myc, FLAG, a sequence of histidineresidues, heamaglutin A, V5, Xpress or GST. Most of these tags can beincorporated directly into the sequence, for instance through PCRamplification by incorporating the appropriate coding sequence in one ofthe PCR amplification primers. However, the tag can also be introducedby other means such as covalent binding of the appropriate nucleic acidsequence encoding the tag moiety with the 3′ or 5′ end of the nucleicacid sequence encoding the polypeptide sequence. This is the case forGST.

Purification of the recombinant secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptides according to the present invention is then carriedout by passage onto a nickel or copper affinity chromatography column,such as a Ni NTA column or a Q-Sepharose column.

In another embodiment of the above method, the polypeptide thus producedis further characterized, for example by binding onto an immuno-affinitychromatography column on which polyclonal or monoclonal antibodiesdirected to the secreted soluble α₂δ-2 subunit polypeptide of interesthave been previously immobilised.

In another embodiment of the invention, the secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide can be only partially purified. Forinstance, it can be purified along with other contaminating proteinsusing an appropriate chromatography matrix such as an ion-exchangechromatography column. In such instances, it is not required to tag thedesired polypeptide of interest.

The most preferred embodiment contemplated by the inventors concerns theuse of a purified tagged secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide. A particularly preferred tag is a nucleotide sequenceencoding from 2 to 10, and preferably 6 histidine residues.

With regard to the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide used subsequently in the screening assay of the invention,several possibilities are also open to the skilled person.

In a first and preferred embodiment, the secreted soluble α₂δ-2, α₂δ-3or α₂δ-4 subunit polypeptide comprises a tag moiety which can beselected among the tags referred to above. Such tagged polypeptides areparticularly useful in SPA or flashplate assays. A preferred tag is thenucleotide sequence encoding histidine residues referred to above.

In a second embodiment, the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide can be used without a tag. This is the case forinstance in SPA or flashplate assays comprising beads or plates coatedwith wheat germ lectin. In such an embodiment, the tag is not needed asthe carbohydrate moieties of the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide bind directly to the wheat germ lectin-coated beadsor plates.

1) Labelled Compounds which Bind the Secreted Soluble α₂δ-2, α₂δ-3 orα₂δ-4 Subunit Polypeptide

In cases where the α₂δ-2, α₂δ-3 or α₂δ-4 binding site is the[³H]gabapentin binding site, the preferred labelled compound which canbe used is of course gabapentin itself. However, gabapentin is not theonly labelled compound which can be used in this context. Indeed, it hasbeen previously demonstrated that saturation binding analyses on porcinesynaptic plasma cerebral cortex membranes performed in the presence ofL-leucine indicate a competitive interaction of the amino acid with the[³ H]gabapentin binding site, significantly reducing [³H]gabapentinbinding affinity for the site. The inventors believe that thiscompetitive interaction is true across across all the amino-acids listedin table 1 below. TABLE 1 Binding affinities of selected amino acids(IC₅₀ < 500 nM) for the [³H]gabapentin site in porcine corticalmembranes COMPOUND IC₅₀ (NM) ARITHMETIC MEAN (N = 3) ± S.E.M. Gabapentin42.1 ± 5.5 L-Norleucine 23.6 ± 6.7 L-Allo-Isoleucine 32.8 ± 6.0L-Methionine  49.6 ± 10.0 L-Leucine  61.3 ± 20.9 L-Isoleucine 68.8 ± 1.9L-Valine 330 ± 18 L-Phenylalanine 351 ± 89

It is therefore possible to use commercialy available labelled forms ofthese high affinity ligands in replacement of gabapentin. The utility of[³H]L-leucine has been demonstrated in a filter binding assay and in aflashplate assay format. The inventors believe that labelled amino acidsbut also other compounds, with affinities preferably below 500 nM in thebinding assay can be used as replacements of gabapentin.

With regard to the label, several embodiments can be used in the contextof the invention. Preferred labels are of course radioactive labels, alist of which is provided further in this specification.

2) Assay Formats and Conditions

Several assay formats can be used to carry out the method of the presentinvention. Preferred assay formats include scintillation assays such asthe scintillation proximity assay (SPA) or the flashplate assay. Otherassay formats well known to those skilled in the arts such as the filterbinding assay and the centrifugation assay are also contemplated in thepresent invention.

SPA and flashplate assays are preferred assay formats for the presentinvention. Additional details on these assays are provided below.

Scintillation Assay Format

Scintillation assays technology either involves the use of scintillantbeads (for the SPA assay) or plates (for the flashplate assay). SPAbeads are usually made from either cerium-doped yttrium ion silicate(y2SiO5:Ce) or polyvinyltoluene (PVT) containing an organic scintillantsuch as PPO. Flashplates commonly used are those such as Ni chelateflashplates although other flashplates can also be used.

Assays are usually carried out in aqueous buffers using radioisotopessuch as ³H, ¹²⁵I, ¹⁴c, ³⁵S or ³³P that emit low-energy radiation, theenergy of which is easily dissipated in an aqueous environment. Forexample, the electrons emitted by ³H have an average energy of only 6keV and have a very short path length (−1 ˜tm) in water. If a moleculelabelled with one of these isotopes is bound to the bead or flashplatesurface, either directly or via interaction with another moleculepreviously coupled to the bead or flashplate, the emitted radiation willactivate the scintillant and produce light. The amount of lightproduced, which is proportional to the amount of labelled moleculesbound to the beads, can be measured conveniently with a liquidscintillation (LS) counter. If the labelled molecule is not attached tothe bead or a flashplate surface, its radiation energy is absorbed bythe surrounding aqueous solvent before it reaches the bead, and no lightis produced. Thus, bound ligands give a scintillation signal, but freeligands do not, and the need for a time-consurning separation step,characteristic of conventional radioligand binding assays, iseliminated. The manipulations required in the assays are reduced to afew simple pipetting steps leading to better precision andreproducibility.

The conditions under which SPA and flashplate assays are performed inthe context of the present invention are provided below.

Scintillation Assay Conditions

a) SPA Assay

The SPA assays is first developed to optimize the conditions under whichthe radioligand binds the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide. The parameters which can be varied to optimize radioligandbinding in a typical SPA assay using Amersham beads include assaytemperature, α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide interaction withthe radioligand and the SPA beads, radioligand concentration as well aspH variations.

The temperature at which the assay can be carried out can vary from 1 to30° C. Preferred temperatures range from 18 to 23° C., with 21° C. beingthe most preferred temperature. The interaction of the α₂δ subunitpolypeptide with the SPA beads can be optimized by adjusting theconcentration of the polypeptide and by introducing a reagent which willfavor this interaction. When 50 mg of Amersham SPA beads are used, theα₂δ-1 subunit polypeptide concentration may vary from 0.1 to 10 pmolesper well, with the optimal concentration being generally around 5 to 6pmoles per well.

As for the reagent favoring the interaction between the secreted solubleα₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide and the radioligand as well asthe Amersham SPA beads, the inventors found that imidazole could beefficiently used for that purpose when the α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide is tagged with an amino acid sequence including 6 histidineresidues. Furthermore, and more importantly, it was found that imidazolealso enhanced binding of the radioligand to the α₂δ-2, α₂δ-3 or α₂δ-4polypeptide.

The concentration of the radioligand is evaluated with respect to theconcentration of secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide present in the assay medium. Generally, the concentration ofradioligand varies from 1 nM to 100 nM. A preferred [³H]gabapentinconcentration is about 5 to 20 nM, with a most preferred concentrationbeing about 10 nM. A preferred [³H]leucine concentration is also about 5to 20 nM, with a most preferred concentration being about 10 nM. It isto be noted that the concentration of other radioligands havingaffinities similar to those of [³H]gabapentin and [³H]leucine shouldalso be in the range of about 5 to 20 nM.

Once the optimal radioligand binding conditions have been determined, atest ligand can be introduced in the assay medium to evaluate the levelof displacement of the radioligand. The concentration of test ligand tobe introduced in the assay medium usually varies from 0.1 nM to about100 uM. A preferred test ligand concentration of about 10 uM is usuallya starting point in a high throughput screening assay. Then, dependingon the number of hits obtained, it may be lowered or increased.

It is to be noted that the parameters set forth above, which have beenevaluated for a typical SPA assay using Amersham SPA beads can beadjusted by the skilled person, for example if SPA beads of a differenttype are used.

b) Flashplate Assay

Similarly to the SPA assays, the flashplate can first be developed inorder to optimize the conditions under which the radioligand binds theα₂δ subunit polypeptide. The parameters which can be varied to optimizeradioligand binding in a typical flashplate assay using NEN Ni chelateflashplates also include assay temperature, secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide interaction with both the radioligandand the flashplates, radioligand concentration as well as pH variations.

The temperature at which the assay can be carried out can vary from 1 to30° C. Preferred temperatures range from 18 to 23° C., with 21° C. beingthe most preferred temperature.

The interaction of the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide with the flashplates can be optimized by adjusting theconcentration of the polypeptide and by introducing a reagent which willfavor this interaction. When a standard NEN Ni chelate flashplate isused, the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptidevolume usually varies between 0.5 and 20 ul for a concentration ofsecreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide of 0.6pmol/ul. As the published maximum binding capacity of NEN p plates isabout 6 pmol per well, the inventors consider that an optimalconcentration of secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunitpolypeptide is probably around 5 pmol per well at 8 ul.

With regard to the reagent favoring the interaction between the secretedsoluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide and the radioligand aswell as the flashplates, the inventors believe that imidazole could alsobe efficiently used for that purpose when the secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide is tagged with an amino acid sequenceincluding 6 histidine residues. The inventors also believe thatimidazole concentrations can substantially enhanced binding of theradioligand to the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 polypeptide.The optimal concentration of imidazole used to enhance radioligandbinding varies depending on the concentration of secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide used in the assay. For instance, whenthe volume of the α₂δ-1 subunit polypeptide is about 10 ul ul (α₂δ-2,α₂δ-3 or α₂δ-4 polypeptide concentration of 0.6 pmol/ul), the optimalimidazole concentration can vary between 1 and 20 mM, with aconcentration of about 10 mM being preferred. As mentioned previously,other compounds such as histidine as well as pH variations may be usedto enhance radioligand binding.

The concentration of the radioligand is evaluated with respect to theconcentration of α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide present inthe assay medium. Generally, the concentration of radioligand variesfrom 1 nM to 100 nM. A preferred [³H]gabapentin concentration is about 5to 20 nM, with a most preferred concentration being about 10 nM. Apreferred [³H]leucine concentration is also about 5 to 20 nM, with amost preferred concentration being about 10 mM. It is to be noted thatthe concentration of other radioligands having affinities similar tothose of [³H]gabapentin and [³H]leucine should also be in the range ofabout 5 to 20 nM.

Once the optimal radioligand binding conditions have been determined, atest ligand can be introduced in the assay medium to evaluate the levelof displacement of the radioligand. The concentration of test ligand tobe introduced in the assay medium usually varies from 0.1 nM to about100 uM. A preferred test ligand concentration of about 10 uM is usuallya starting point in a high throughput screening assay. Then, dependingon the number of hits obtained, it may be lowered or increased.

The inventors have tested the displacement of a particular radioligand,[³H]gabapentin, with (S+)-3-isobutly gaba. The data provided in theexamples which follow clearly shows that the assay can be used in highthroughput competition studies.

E) Purified Recombinant Secreted Soluble α₂δ-2, α₂δ-3 or α₂δ-4Polypeptides

Another object of the present invention consists of a purified orisolated recombinant polypeptide comprising the amino acid sequence of asecreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide.

Preferred isolated recombinant polypeptides of the invention includethose having at least 80%, preferably 90%, more preferably 95, and mostpreferably 98 or 99%, amino-acid identity with polypeptides comprisingfrom amino acid 1 to between amino-acids 1027 and 1145, preferablybetween amino-acids 1062 and 1145 of SEQ ID No20, as well as thosehaving at least 80%, preferably 90%, more preferably 95, and mostpreferably 98 or 99%, amino-acid identity with polypeptides comprisingfrom amino acid 1 to between amino-acids 984 and 1085, preferablybetween amino-acids 1019 and 1085 of SEQ ID No22.

In a further preferred embodiment, the polypeptide comprises an aminoacid sequence having at least 80%, preferably 90%, more preferably 95%,and most preferably 98% or 99% amino acid identity with the amino acidsequence of SEQ ID No4, SEQ ID No5, SEQ ID No6, SEQ ID No10, SEQ IDNo11, SEQ ID No12, SEQ ID No16, SEQ ID No17 and SEQ ID No19.

F) Modified Secreted Soluble α₂δ-2, α₂δ-3 or α₂δ-4 Subunit Polypeptides

The invention also relates to secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide comprising amino acid changes ranging from 1, 2, 3,4, 5, 10, 20, 25, 30, 35, 40 substitutions, additions or deletions ofone amino acid as regards to polypeptides of anyone of the amino acidsequences of the present invention. Preferred sequences are those of SEQID No4, SEQ ID No5, SEQ ID No6, SEQ ID No10, SEQ ID No11, SEQ ID no12,no16, SEQ ID no17 and SEQ ID No18.

In the case of an amino acid substitution in the amino acid sequence ofa polypeptide according to the invention, one or several consecutive ornon-consecutive amino-acids are replaced by “equivalent” amino-acids.The expression “equivalent” amino acid is used herein to designate anyamino acid that may be substituted for one of the amino-acids belongingto the native protein structure without decreasing the bindingproperties of the corresponding peptides to the antibodies raisedagainst the polypeptides of the invention. In other words, the“equivalent” amino-acids are those which allow the generation or thesynthesis of a polypeptide with a modified sequence when compared to theamino acid sequence of the secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptides of interest, said modified polypeptide being ableto bind to the antibodies raised against the secreted soluble α₂δ-2,α₂δ-3 or α₂δ-4 subunit polypeptide of interest and/or to induceantibodies recognizing the parent polypeptide.

Alternatively, amino acid changes encompassed are those which will notabolish the biological activity of the resulting modified polypeptide.These equivalent amino-acids may be determined either by theirstructural homology with the initial amino-acids to be replaced, by thesimilarity of their net charge or of their hydrophobicity, andoptionally by the results of the cross-immunogenicity between the parentpeptides and their modified counterparts.

The peptides containing one or several “equivalent” amino-acids mustretain their specificity and affinity properties to the biologicaltargets of the parent protein, as it can be assessed by a ligand bindingassay or an ELISA assay.

Examples of amino-acids belonging to specific classes include Acidic(Asp, Glu), Basic (Lys, Arg, His), Non-polar (Ala, Val, Leu, Ile, Pro,Met, Phe, Trp) or uncharged Polar (Gly, Seu, Thr, lys, Tyr, Asn, Gln)amino-acids.

Preferably, a substitution of an amino acid in α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide of the invention, or in a peptide fragment thereof,consists in the replacement of an amino acid of a particular class foranother amino acid belonging to the same class.

By an equivalent amino acid according to the present invention is alsocontemplated the replacement of a residue in the L-form by a residue inthe D form or the replacement of a Glutamic acid (E) residue by aPyro-glutamic acid compound. The synthesis of peptides containing atleast one residue in the D-form is, for example, described by Koch(1977).

A specific embodiment of a modified peptide of interest according to thepresent invention, includes, but is not limited to, a peptide molecule,which is resistant to proteolysis. This is a peptide in which the —CONH—peptide bond is modified and replaced by a (CH₂NH) reduced bond, a(NHCO) retro inverso bond, a (CH₂—O) methylene-oxy bond, a (CH₂S)thiomethylene bond, a (CH₂CH₂) carba bond, a (CO—CH₂) cetomethylenebond, a (CHOH—CH₂) hydroxyethylene bond), a (N—N) bound, a E-alcene bondor also a —CH═CH-bond.

The invention also encompasses secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4subunit polypeptide in which at least one peptide bond has been modifiedas described above.

The polypeptides according to the invention may also be prepared by theconventional methods of chemical synthesis, either in a homogenoussolution or in solid phase. As an illustrative embodiment of suchchemical polypeptide synthesis techniques, it may be cited thehomogenous solution technique described by Houbenweyl (1974).

The secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide ofinterest, or a fragment thereof may thus be prepared by chemicalsynthesis in liquid or solid phase by successive couplings of thedifferent amino acid residues to be incorporated (from the N-terminalend to the C-terminal end in liquid phase, or from the C-terminal end tothe N-terminal end in solid phase) wherein the N-terminal ends and thereactive side chains are previously blocked by conventional groups.

For solid phase synthesis, the technique described by Merrifield (1965a;1965b) may be used in particular.

G) Antibody Production

The secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptides of theinvention and their peptide fragments of interest can be used for thepreparation of antibodies.

Polyclonal antibodies may be prepared by immunization of a mammal,especially a mouse or a rabbit, with a polypeptide according to theinvention that is combined with an adjuvant of immunity, and then bypurifying the specific antibodies contained in the serum of theimmunized animal on an affinity chromatography column on which haspreviously been immobilized the polypeptide that has been used as theantigen.

Monoclonal antibodies may be prepared from hybridomas according to thetechnique described by Kohler and Milstein (1975).

The present invention also deals with antibodies produced by the triomatechnique and by the human B-cell hybridoma technique, such as describedby Kozbor et al. (1983).

Antibodies of the invention also include chimeric single chain Fvantibody fragments (U.S. Pat. No. 4,946,778; Martineau et al., (1998),antibody fragments obtained through phage display libraries Ridder etal. (1995) and humanized antibodies (Leger et al., (1997)).

H) Screening Assays

The invention concerns a method for the screening of ligands which bindsoluble secreted α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide. Moreparticularly, the targeted α₂δ-2, α₂δ-3 or α₂δ-4 subunit binding site ispreferably the [³H]gabapentin binding site. The various parameters ofthe method of the invention are described in further detail below.

EXAMPLES Example 1 Construction of a Nucleotide Sequence Encoding aSoluble Secreted Human α₂δ-2 Subunit Polypeptide Deletion Mutant of SEQID No23

a) Primer Design

PCR primers were designed to generate the secreted soluble human α₂δ-2deletion mutant of SEQ ID No 23 as follows:

-   -   5′ PCR primer: This was designed to engineer in a KOZAK        translation initiation consensus sequence prior to the coding        sequence (Kozak JBC 266 19867-19870)    -   3′ PCR primer: This was designed to engineer in six histidine        residues followed by a stop-codon at the desired location in the        coding sequence. In addition to the stop codon the α₂δ-2 primers        also included an Eco RI restriction site.

The bold region in each primer sequence denotes the ‘tagged’ region;addition of sequences not present in the template. Primers were customsynthesized by Perkin Elmer Applied Biosystems UK to the ABI ready puregrade, supplied lyophilized then resuspended to 15 μM in 10 mM TE. JB197and 198 were provided with 5′ phosphate groups: 5′ Primer JB197(5′-TCGCCACCATGGCGGTGCCGGCTC-3′, SEQ ID NO 25) 3′ Primer JB198(5′-TCGGAATTCCTCAGTGATGGTGATGGTGATGGGCCCCGCGGCCACAGTC-3′, SEQ ID NO 28)b) Protocol for PCR Mediated 5′ Kozak and 3′ 6His Tagging of Human α₂δ-2

The full length human α₂δ-2 gene (Gen Bank Accession Number AF042792) ina pcDNA 3 vector as described in Brown, J. P. and Gee, N. S., Cloningand deletion mutagenesis of the α₂δ calcium channel subunit from porcinecerebral cortex, The journal of biological chemistry,273(39):25458-25465) was used as the template in the following PCRreaction. The reagents were added in the following order in triplicateto a 96 well PCR plate: μl 10x Pfx Amplification buffer 5 10 mM dNTPs1.5 50 mM MgSO₄ 1 15 μM JB197 1.5 15 μM JB198 1.5 100 ng/μlpcDNA3.1-humans-α₂δ-2 1 10x PCR Enhancer 5 H₂O 32.7 2.5 UNITS/μL PFXPOLYMERASE 0.8 μLThe plate was the cycled on an MJ Tetrad DNA engine according to thefollowing cycling conditions:

-   -   94° C./2 mins

followed by:

-   -   for 30 cycles 94° C./45 sec        -   58° C./45 sec        -   68° C./4 mins

followed by:

-   -   68° C./10 mins

followed by:

-   -   hold at 4° C.

The 3366 bp product was then gel purified from a 1% TAE agarose gelusing QIAEX beads and eluted in approximately 50 μl TE.

Example 2 Cloning of the PCR Fragments of Example 1 into the BaculovirusTransfer Vector pFastBac1

The PCR products of Example 1 were cloned into Stu I digested, calfintestinal phosphatase dephosphorylated, phenol chloroform extracted andQIAEX gel purified pFastBac1 (Life Technologies) using the Rapid DNAligation kit (Roche Diagnostics) transforming XL1-blue (α₂δ-1b) E. Colicells:

a) Screening for Positive Recombinants

Given that the PCR product was cloned by blunt-end ligation a screen wasrequired to select a recombinant with the gene ligated in the positiveorientation with respect to the polyhedrin promoter in pFastBac1. Thiswas achieved by restriction digest of miniprep DNA (Qiagen miniprep kit)prepared from colony minicultures and analysis on a 1% TAE agarose gel.A positive clone was identified according to the following digestpatterns:

-   -   SEQ ID No 23 in pFastBac1    -   Eco RI digest performed on miniprep DNA        -   Predicted fragments (bp)    -   PCR product cloned in a positive orientation 4773 and 3368    -   PCR product cloned in a negative orientation 8127 and 14        b) Sequencing Analysis of Selected Clones

One positive was selected for this clone and used to prepare a plasmidDNA stock of the desired construct (QIAGEN maxi kit). Confirmatorysequence reactions were performed using the Big Dye terminatorsequencing kit and run on an ABI 310 Prism Genetic Analyzer. Sequenceanalysis of both coding strands was performed using a selection ofsequencing oligonucleotide primers.

Example 3 Protocol for Establishing Baculovirus Banks for the Expressionof the α₂δ-2 Deletion Mutant SEQ ID No23

Essentially, the protocol used to generate the baculovirus banks is thatoutlined in the Life Technologies Bac-to Bac™ baculovirus expressionsystems manual.

a) Transposition of DH10Bac E Coli Cells

One ng (5 μl) of the recombinant pFastBac-1 construct containing thenucleotide sequence encoding the porcine α₂δ-2 deletion mutant of SEQ IDNo23 was added to 100 μl of DH10Bac cells thawed on ice. The cells werethen mixed gently by tapping the tube then incubated on ice for 30minutes before heat shock treatment by incubation in a 42° C. water bathfor 45 seconds. The mixture was then chilled on ice for 2 minutes beforethe addition of 900 μl of S.O.C. medium. The mixture was then placed ina shaking incubator (200 rpm) at 37° C. for 4 hours. The cells were thenserially diluted (10 fold dilutions from 10⁻¹ to 10³) and 10 μl of eachdilution plated on LB agar plates containing 50 μg/ml kanamycin, 7 μg/mlgentamicin, 10 μg/ml tetracycline, 100 μg/ml Bluo-gal and 40 μg/ml IPTG.The plates were incubated at 37° C. for between 1 and 3 days untildiscrete colonies of blue and white colour were discernible.

b) Isolation of Recombinant DNA

White colonies (containing the recombinant bacmid) were picked and grownfor 24 hours (to stationary phase) at 37° C. with shaking (200 rpm) in 2ml of LB containing 50 μg/ml kanamycin, 7 μg/ml gentamicin and 10 μg/mltetracycline. 1.5 ml of culture was then transferred to a microfuge tubeand centrifuged at 14,000×g for 1 minute. The supernatant was removedand the cells resuspended gently in 0.3 ml of 15 mM Tris-HCl (pH8.0), 10nM EDTA, 100 μg/ml RNase A. 0.3 ml of 0.2N NaOH, 1% SDS was then addedand the mixture mixed gently before incubation at 22° C. for 5 minutes.Then 0.3 ml of 3M Potassium acetate (pH5.5) was added and the sampleplaced on ice for 10 minutes. After centrifugation at 14,000×g for 10minutes the supernatant was transferred to a tube containing 0.8 ml ofisopropanol, mixed then placed on ice for 10 minutes beforecentrifugation at 14,000×g for 10 minutes. The supernatant was thendiscarded and the pellet rinsed with 0.5 ml of 70% ethanol beforecentrifugation at 14,000×g for 5 minutes. This 70% ethanol rinse wasthen repeated before removing all of the supernatant and air drying thepellet for 10 minutes at room temperature. The pellet was finallyresuspended in 40 μl of TE.

c) Transfection of sf9 Cells with the Recombinant Bacmid DNA

A 6-well tissue culture plate was seeded with 0.9×10⁶ sf9 cells (cellsat log phase having grown from a culture passaged at 0.3×10⁶ cells/ml)per 35 mm well in 2 ml of Sf-900 II SFM media containing 50 units/mlpenicillin and 50 μg/ml streptomycin. Cells were left to attach at 27°C. for 1 hour. Bacmid DNA prepared as described above (5 μl) was addedto 200 μl of Sf-900 II SFM media containing 6 μl of CELLFECTIN and mixedbefore incubation at room temperature for 45 minutes. The cells werewashed once with 2 ml of Sf-900 II SFM media without antibiotics then0.8 ml of Sf-900 II SFM media was added to each tube containing thelipid-DNA complex. The wash buffer was removed from the cells and the 1ml of diluted lipid-DNA complex overlaid on the cells. The cells wereincubated for 5 hours at 27° C. after which time the transfectionmixture was removed and 2 ml of Sf-900 II SFM media containing 50units/ml penicillin and 50 μg/ml streptomycin was added. The cells werethen incubated for 72 hours.

After incubation for 72 hours the media was removed from the cells andcentrifuged at 500×g for 5 minutes. The supernatant was then transferredto a fresh tube, this was labelled as the P0 bank and stored at 4° C. inthe dark. The P1 bank was prepared by passaging sf9 cells at approx5×10⁶ cells/ml to 2×10⁶ cells/ml (100 ml in a 250 ml Erlenmeyer flask)and adding 0.5 ml of the P0 bank harvested above. The cells were thenincubated shaking (200 rpm) at 27° C. for 4 days. Under sterileconditions the culture was centrifuged at 500×g for 10 minutes and thesupernatant 0.2 μM filtered (P1 bank). The P2 bank was prepared byadding 2 ml of P1 bank per 400 ml culture (in 1 L Erlenmeyer flasks)passaged as above to 2×10⁶ cells/ml. The culture was incubated as beforefor 4 days and the supernatant harvested and filtered as described forthe P1 bank. The supernatant was first pooled then aliquoted (10 ml) andstored at 4° C.

Example 4 Expression of the a28-2 Deletion Mutant of SEQ ID No23

To sf9 cells passaged from ˜5×10⁶ cells/ml to 2×10⁶ cells/ml in Sf-900II SFM media was added 0.1 ml virus per 100 ml of cells of theappropriate viral bank (400 ml volumes in 1 L Erlenmeyer flasks). Thecells were then cultured for 4-5 days at 27° C. with 110 rpm shaking.Expression of the protein was confirmed by SDS-PAGE and Western blottingusing an anti penta-His monoclonal antibody (Qiagen) and was detected inthe culture supernatant and cell lysate.

Example 5 Purification of α₂δ-2Deletion Mutant of SEQ ID No23

The-α₂δ-2 deletion mutant of SEQ ID No23 was purified from the celllysate following the purification strategy outlined below:

The culture was centrifuged at 6,000×g for 10 minutes and thesupernatant removed. The weight of the cell pellet was determined beforere-suspension in 20 mM Tris pH8.0, 100 mM KCl, 1% P40-Nonidet (100 mlper 20 g of wet cells). A protease inhibitor cocktail (Sigma, Cat #P8849), 1 ml/L, was added to the mixture. The solution was then stirredfor 10 minutes before centrifugation for 1 hour at 30,000×g and 4° C.The supernatant was concentrated (30 kDa cut off) to approx. ˜300 mlthen centrifuged for 1 hour at 100,000×g.

Supernatant containing the soluble proteins was diluted 1:3 in 10 mMTris-HCl pH8.0 (equilibration buffer) and loaded onto a pre-equilibratedQ-Sepharose column (2.5 cm i.d.×30 cm h.) at a flow rate of 900 ml/h.After washing with equilibration buffer until a stable A₂₈₀ baseline hadbeen achieved, protein was eluted with 20 mM Tris-HCl pH8.0, 0.5M KCl,10 mM Imidazole.

The eluate was then loaded onto a Ni-NTA (Qiagen) column (2.5 cm i.d.×6cm h.) pre-equilibrated in 20 mM Tris pH8.0, 0.5M KCl, 10 mM Imidazoleat a flow rate of 2 ml/min. The column was washed successively withbuffer A (20 mM Tris pH8.0, 0.5M KCl, 20 mM Imidazole), buffer B (100 mMTris-HCl pH8.0, 1 M KCl), and buffer A again. Elution was performed withbuffer C (20 mM Tris-HCl pH8.0, 100 mM KCl, 0.5M Imidazole). The Ni—NTAeluate (˜50 ml) was concentrated (30 kDa cut-off) to ˜2 ml and appliedat 1 ml/min and in 0.2 ml aliquots, to an FPLC Superdex-200 columnequilibrated in 10 nM HEPES, pH7.4, 150 mM NaCl. Fractions containingthe polypeptide of SEQ ID No23 were pulled.

Example 6 SPA assay of [³H[gabapentin Binding to the Secreted SolubleHumanα₂δ-2 Subunit of SEQ ID No23

The assay is carried out at 21° C. Assay components are added in thefollowing order (all reagents are diluted in 10 mM HEPES (pH 7.4 at 21°C.) to 96-well Optiplates:

-   -   25 μl imidazole at various concentrations (diluted from a 1 M        stock pH8.0, see assay details)    -   50 μl 10 mM HEPES pH 7.4    -   25 μl (50 mg) SPA beads (Amersham)    -   100 μl s-α₂δ-2 subunit polypeptide of SEQ ID No 23 (2 μl protein        diluted to 100 μl)    -   25 μl radioligand ([³H]gabapentin obtained from example 5

Immediately after adding radioligand, the optiplates were loaded in thePackard Top Count scintillation counter to follow the binding timecourse. Imidazole was first used in the assay to optimize the specificinteraction of the protein's 6His tag with the SPA bead. Imidazoleitself (up to 100 mM) in the filtration assay has no effect on[³H]gabapentin binding (n=1).

Example 7 Ni Flashplate Assay of [³H]gabapentin Binding to SecretedSoluble Human α₂δ-2 (SEQ ID No23)

Assays are carried out at 21° C. in a final volume of 250 μl in 96-wellNEN Ni chelate flash plates. Assay components are added in the followingorder (all reagents were diluted in 10 mM HEPES (pH 7.4 at 21° C.)):

-   -   25 μl 10 mM HEPES pH7.4    -   25 μl imidazole at various concentrations (diluted from a 1M        stock pH8.0, see assay details)    -   75 μl 10 mM HEPES pH 7.4    -   100 μl s-α₂δ-2-6His (2 μl protein diluted to 100 μl) obtained        from example 5    -   25 μl radioligand ([³H]gabapentin (65 Ci/mmole)

Immediately after adding the radioligand, flash plates are loaded in thePackard Top Count scintillation counter to follow the binding timecourse. The ‘[³H] flash plate’ programme (cpm) is used to monitoractivity. Imidazole is first used in the assay to optimize the specificinteraction of the protein's 6His tag with the Ni flashplate.

Example 8 Ni Flashplate Assay of [³H]Leucine Binding to Secreted SolubleHuman α₂δ-2-6His

The procedure described in example 7 is repeated, except that[³H]gabapentin is replaced by 25 ul (10.1 nM) of [³H]Leucine (141Ci/mmole).

Example 9 Ni Flashplate Assay Studying Competitive Binding of[³H]gabapentin and (S+)-3-isobutyl GABA to Human α₂δ-2-6His (SEQ IDNo23).

Assays are carried out at 21° C. in a final volume of 250w1 in 96-wellNEN Ni chelate flash plates. Wells are set up for both ‘total’ and‘non-specific’ binding. Specific binding is defined as that remainingafter subtraction of the average of the ‘non-specific binding’ valuesfrom the average of the ‘total’ binding values. Assay components areadded in the following order (all reagents were diluted in 10 mM HEPES(pH 7.4 at 21° C.)):

-   -   25 μl 10 mM HEPES pH7.4 or 25 ul othe test compound at the        appropriate concentration in HEPES    -   25 μl 200 mM imidazole (diluted from a 1M stock pH8.0, see assay        details)

Total binding 75 μl 10 mM HEPES pH 7.4

Non-specific binding 50 μl 10 mM HEPES pH 7.4 and 25 μl 100 μl M(S+)-3-isobutyl GABA

-   -   100 μl α₂δ-2-6His (2 μl protein* diluted to 100 μl)    -   25 μl radioligand ([³H]gabapentin or [³H]Leucine)        The source of α₂δ-2-6His is that purified by fplc Superdex-200        gel filtration (see example 5)

Immediately after adding radioligand, flash plates are loaded in thePackard Top Count scintillation counter to follow the binding timecourse. Incubation time before the assay is 3 hours. The ‘[3H] flashplate’ programme (cpm) is used to monitor activity Competition studiesare compared across the flash-plate and filter binding methodologies inorder to validate the new assay technology with the established filterbinding methodology.

GraphPad Prism software is used to process competition curve data anddetermine IC₅₀ and hill slope values. Twelve point competition curveswith half log dilution steps of test compounds are used in theexperiments.

Example 10 Filter Binding Assay of [³H]gabapentin Binding to theRecombinant Polypeptide of SEQ ID No23

Assays were carried out at 21° C. in a final volume of 250 μl in 96-deepwell plates. Assay components were (all reagents were diluted in 10 mMHEPES (pH 7.4 at 21° C.)):

-   -   25 μl compound to test    -   200 μl Polypeptide of SEQ ID No23 (3 μl protein diluted to 200        μl)    -   25 μl radioligand ([³H]gabapentin (65 Ci/mmole)

Plates were incubated at room temperature for 1 h prior to filtering onto 96-well GF/B Unifilter plates pre-soaked in 0.3% polyethylenimine.Filters were washed with 3×1 ml 50 mM Tris-HCl (pH 7.4 at 4° C.), anddried over-night. Scintillant (Microscint O, 50 μl) was added and theplates counted using a Packard Top Count scintillation counter. Specificbinding was ˜98% of the ‘total’ value. In [³H]gabapentin saturationstudies, the K_(D) (nM) obtained was about 10.62. Table 2 below providesthe results of the competition studies.

Example 11 Construction of a Nucleotide Sequence Encoding a SolubleSecreted Mouse α₂δ-3 Deletion Mutant of SEQ ID No25 as follows.

a) Primer Design

PCR primers were designed to generate the secreted soluble mouse α₂δ-3deletion mutant of SEQ ID No 25 as follows:

5′ PCR primer: This was designed to engineer in a KOZAK translationinitiation consensus sequence prior to the coding sequence (Kozak JBC266 19867-19870)

3′ PCR primer: This was designed to engineer in six histidine residuesfollowed by a stop-codon at the desired location in the coding sequence.In addition to the stop codon the α₂δ-3 primers also included an Eco RIrestriction site.

The bold region in each primer sequence denotes the ‘tagged’ region;addition of sequences not present in the template. Primers were customsynthesized by Perkin Elmer Applied Biosystems UK to the ABI ready puregrade, supplied lyophilized then resuspended to 15 μM in 10 mM TE. JB201and 202 were provided with 5′ phosphate groups: 5′ Primer JB201(5′-TCGCCACCATGGCCGGGCCGGGC-3′, SEQ ID NO 27) 3′ Primer JB202(5′-TCTCAGTGATGGTGATGGTGATGCGATGCACCCCCACACTCTC-3′, SEQ ID NO 28)b) Protocol for PCR Mediated 5′ Kozak and 3′ 6His Tagging of Mouse α₂δ-3

The full length mouse α₂δ-3 gene (Gen Bank Accession number AJ010949) inthe pcDNA3 vector as described in Brown, J. P. and Gee, N. S., Cloningand deletion mutagenesis of the α₂δ calcium channel subunit from porcinecerebral cortex, The journal of biological chemistry,273(39):25458-25465) was used as the template in the following PCRreaction.

The reagents were added in the following order in triplicate to a 96well PCR plate: μl 10x Pfx Amplification buffer 5 10 mM dNTPs 1.5 50 mMMgSO₄ 1 15 μM JB201 1.5 15 μM JB202 1.5 100 ng/μl pcDNA3-mouse-α₂δ-3 110x PCR Enhancer 5 H₂O 32.7 2.5 UNITS/μL PFX POLYMERASE 0.8 μL

The plate was the cycled on an MJ Tetrad DNA engine according to thefollowing cycling conditions:

-   -   94° C./2 mins

followed by:

-   -   for 30 cycles 94° C./45 sec        -   60° C./45 sec        -   68° C./4 mins

followed by:

-   -   68° C./10 mins

followed by:

-   -   hold at 4° C.

The 3244 bp product was then gel purified from a 1% TAE agarose gelusing QIAEX beads and eluted in approximately 50 μl.

The truncated protein of SEQ ID No24 was expressed such the procedure ofexample 2,3 and 4.

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1-7. (canceled)
 8. A method for the amplification of a nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel α₂δ-n subunit polypeptide wherein n is 2, 3 or 4, said method comprising the steps of: (a) contacting a test sample suspected of containing the target secreted soluble α₂δ-n subunit nucleic acid, or a sequence complementary thereto, with an amplification reaction reagent comprising a pair of amplification primers located on either side of the α₂δ-n subunit nucleic acid region to be amplified, and (b) optionally, detecting the amplification products. 9-12. (canceled)
 13. A purified or isolated recombinant polypeptide comprising the amino acid sequence of a secreted soluble α₂δ-2, α₂δ-3 or α₂δ-4 subunit polypeptide.
 14. A recombinant polypeptide according to claim 14, having at least 80% amino-acid identity with a polypeptide comprising: from amino acid 1 to between amino acids 1027 and 1062 of the amino acid sequence of SEQ ID No20, or from amino acid 1 to between amino acids 1019 and 1079 of the amino acid sequence of SEQ ID No22.
 15. A recombinant polypeptide according to claim 14, wherein said recombinant polypeptide is selected from the group consisting of the amino acid sequences of SEQ ID NO:4 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 10 SEQ ID NO:11. SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18
 16. A method for the screening of ligands which bind a cerebral cortical voltage-dependent calcium channel α₂δ-n subunit wherein n is 2, 3 or 4, said method comprising the steps of: contacting a secreted soluble recombinant calcium channel α₂δ-n subunit polypeptide with: a ligand of interest; and a labelled compound which binds the α₂δ-n subunit; and measuring the level of binding of the labelled compound to the α₂δ-n subunit.
 17. A method according to claim 16, wherein said method is a scintillation proximity assay.
 18. A method according to claim 16, wherein said method is a flashplate assay.
 19. A method according to claim 16, wherein said method is a filter binding assay.
 20. A method according to claim 16, wherein said secreted soluble recombinant calcium channel α₂δ-n subunit polypeptide is selected from polypeptides having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 984 and 1063, preferably between amino-acids 994 and 1054, and most preferably between amino-acids 1019 and 1054 of SEQ ID NO:5 or SEQ ID NO:16.
 21. A method according to claim 16, wherein said secreted soluble recombinant calcium channel α₂δ-n subunit polypeptide is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, with the polypeptides of SEQ ID NO: 9 and SEQ ID NO: 15 being most preferred.
 22. A kit for the screening of ligands which bind bind a cerebral cortical voltage-dependent calcium channel α₂δ-n subunit wherein n is 2, 3 or 4, said kit comprising: a secreted soluble recombinant calcium channel α₂δ-n subunit; and a labelled compound which binds to the α₂δ-n subunit. 